Rutaecarpine analogs and applications thereof

ABSTRACT

The present invention provides RUT analogs with various biological activities. In particular, the biological activities comprise anti-inflammatory activity, vasodilator effects, migration/invasion-suppressive activities, ability against damage due to remodeling between the epithelium and endothelium, collagen deposition and cardiac fibrosis suppress, Snail protein inhibitory effect, etc., which may improve cardiac, vasodilation, and lung functions. The RUT analogs disclosed herein also exhibit a lower cytotoxicity comparing to RUT.

FIELD OF THE INVENTION

The present invention relates to rutaecarpine analogs, in particularfluorinated rutaecarpine. The present invention also relates to medicalapplications of rutaecarpine analogs.

BACKGROUND OF THE INVENTION

Endothelial cells (ECs) of arteries are important for the trafficking ofnutrients and participate in many physiologic events, such asinflammation and angiogenesis. Atherosclerosis is primarily associatedwith a series of reactions within the tunica intima and involvesmonocyte recruitment, macrophage formation, lipid accumulation,extracellular matrix (ECM) production, and smooth muscle cell migration.Compounds with inhibitory effects on vascular inflammation and cellmigration would be beneficial for antiatherogenic progression.

Rutaecarpine (RUT) is one of the main bioactive ingredients extractedfrom the traditional medicine Evodia rutaecarpa and can improveatherosclerosis by preventing monocyte adhesion to the vascularendothelium. RUT reduced the prostaglandin production oflipopolysaccharide (LPS)-activated RAW264.7 macrophages, but did notaffect levels of cyclooxygenase (COX)-2 messenger (m)RNA or protein. Thevasorelaxant effect of RUT in isolated mesenteric arteries was reportedto be associated with Ca²⁺ flux activity based on in vivo tests on mice.RUT lowered blood pressure through the endothelial Ca²⁺-nitric oxide(NO)-cGMP pathway to reduce residual muscle tension. The calcitoningene-related peptide (CGRP), a major neurotransmitter produced inperipheral and central neurons, plays a key role in maintainingendothelial homoeostasis. Decreased plasma CGRP levels cause cardiacsusceptibility to ischemia-reperfusion injury, and RUT reverses thatdecrease by stimulating CGRP production. CGRP counteracts angiotensin(Ang) II-induced endothelial progenitor cell senescence by suppressingreactive oxygen species (ROS) and NADPH oxidase.

Activation of transient receptor potential vanilloid type 1 (TRPV1) inECs may protect against cardiovascular diseases such as hypertension andstroke. Release of CGRP by activation of vanilloid receptors plays animportant role in the vasodilation effects of RUT. NO released byactivation of endothelial NO synthase (eNOS) leads to vascularrelaxation mediated by CGRP and TRPV1 stimulation. TRPV1-dependentatheroprotection was demonstrated in mice. RUT was reported to be apotential therapeutic agent for arterial thrombosis because of itsantiplatelet effect in vivo. Alkaloid compounds also showed anticanceractivities by inducing cell cycle arrest or apoptosis in vitro and invivo. RUT showed high toxicity to lymphoblasts and inhibitedATP-dependent efflux pumps in a blood-brain barrier model with porcinebrain capillary ECs, that thus restricts its application in vasculardiseases. A variety of structural modifications of natural products weredesigned and synthesized for better biological applications. RUTderivatives were designed and synthesized to activate TRPV1 for enhancedvasodilator and hypotensive effects. The 14-N atom of RUT is criticalfor its activity. Bromo-rutaecarpine was designed to broaden thepotential for application. However, the bromo-derivative may not bestable enough due to it being more bulky in substitution.

There are still needs for RUT analogs which exhibit very lowcytotoxicity, but maintain the anti-inflammatory activity andTRPV1-upregulating effects.

SUMMARY OF THE INVENTION

The present invention provides RUT analogs with various biologicalactivities. In particular, the biological activities compriseanti-inflammatory activity, vasodilator effects,migration/invasion-suppressive activities, ability against damage due toremodeling between the epithelium and endothelium, collagen depositionand cardiac fibrosis suppress, Snail protein inhibitory effect, etc.,which may improve cardiac, vasodilation, and lung functions. The RUTanalogs disclosed herein also exhibit a lower cytotoxicity comparing toRUT.

Accordingly, the present invention provides a compound having thefollowing Formula (I) as described herein.

The present invention also provides a pharmaceutical compositioncomprising a compound as described herein, or a pharmaceuticallyacceptable ester, salt, or prodrug thereof, together with apharmaceutically acceptable carrier.

The present invention also provides a method of amelioratinginflammation in a subject, comprising a step of administering a compoundas described herein to the subject.

The present invention also provides a method of suppressing nitrogenoxide (NO) release in a subject, comprising a step of administering asdescribed herein to the subject.

The present invention further provides a method of suppressing TNF-αrelease and/or inhibiting cell migration, cell invasion or both in asubject, comprising a step of administering a compound as describedherein to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A thru 1C show the effects of 10-fluoro-2-methoxyrutaecarpine(F-RUT) on nitric oxide (NO) and tumor necrosis factor (TNF)-α releaseby lipopolysaccharide (LPS)-treated (40 ng/mL) RAW264.7 macrophages: (a)NO levels were detected in culture medium using the Griess reaction; (b)TNF-α release in cell supernatants was detected using a mouse TNF-αQuantikine kit; (c) Cell viability upon F-RUT and rutaecarpine (RUT)treatment for 24 h in an MTT assay. Values are expressed as the mean±SE.*p<0.05, **p<0.01.

FIGS. 2A and 2B show the effect of 10-fluoro-2-methoxyrutaecarpine(F-RUT) on inducible nitric oxide synthase (iNOS) and cyclooxygenase(COX)-2 expressions by lipopolysaccharide (LPS)-treated RAW264.7macrophages: (a), and luciferase reporter plasmid-transfectedmacrophages (b). Cells were transfected with 2.5 μg of the pGL4.32[luc2P/NF-κB-RE/Hygro] reporter plasmid, then treated with differentconcentrations of F-RUT and LPS (40 ng/mL) for 24 h. Levels ofluciferase activity were determined as described in Materials andMethods. Values are expressed as the mean±SE of triplicate tests.*p<0.05, **p<0.01 versus LPS treatment.

FIGS. 3A and 3B show the effects of 10-fluoro-2-methoxyrutaecarpine(F-RUT) on migration and invasion: Cell migration (a) and cell invasion(b) were detected following F-RUT treatment for 0-24 h, and photographedwith a microscope (upper panel). The statistical analysis is shown inthe lower panel. (*p<0.05, **p<0.01).

FIG. 4 shows the effects of 10-fluoro-2-methoxyrutaecarpine (F-RUT) ontransient receptor potential vanilloid-type 1 (TRPV-1) expression andendothelial nitric oxide synthase (eNOS) phosphorylation in human aorticendothelial cells (HAECs). The densitometric ratio is indicated.

FIG. 5A thru 5C show the effects of 10-fluoro-2-methoxyrutaecarpine(F-RUT) on ovalbumin (OVA)-challenged mice: (a) BALB/c mice as targetanimals, (b) KLF10 KO mice as target animals, (c) collagen formationaround the bronchus of KLF10 KO mice. Data are representative of threeto five mice per group. Scale bar is 100 μm.

FIG. 6 shows the average thickness of blood-air-barrier in the tests ofKLF10 KO mice.

FIG. 7 shows the ROS inhibition activity in Zebrafish.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs applying that term in context toits use in describing the present invention. The terminology used in thedescription is for describing particular embodiments only and is notintended to be limiting of the invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms in which case each carbon atomnumber falling within the range is provided), between the upper andlower limit of that range and any other stated or intervening value inthat stated range is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

The articles “a” and “an” as used herein and in the appended claims areused herein to refer to one or to more than one (i.e., to at least one)of the grammatical object of the article unless the context clearlyindicates otherwise. By way of example, “an element” means one elementor more than one element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The term “alkyl” refers to a straight or branched hydrocarbon chainradical consisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to fifteen carbon atoms (e.g., C₁-C₁₅alkyl). In certain embodiments, an alkyl comprises one to thirteencarbon atoms (e.g., C₁-C₁₃ alkyl). In certain embodiments, an alkylcomprises one to eight carbon atoms (e.g., C₁-C₈ alkyl). In otherembodiments, an alkyl comprises one to five carbon atoms (e.g., C₁-C₅alkyl). In other embodiments, an alkyl comprises one to four carbonatoms (e.g., C₁-C₄ alkyl). In other embodiments, an alkyl comprises oneto three carbon atoms (e.g., C₁-C₃ alkyl). In other embodiments, analkyl comprises one to two carbon atoms (e.g., C₁-C₂ alkyl). In otherembodiments, an alkyl comprises one carbon atom (e.g., C₁ alkyl). Inother embodiments, an alkyl comprises five to fifteen carbon atoms(e.g., C₅-C₁₅ alkyl). In other embodiments, an alkyl comprises five toeight carbon atoms (e.g., C₅-C₈ alkyl). In other embodiments, an alkylcomprises two to five carbon atoms (e.g., C₂-C₅ alkyl). In otherembodiments, an alkyl comprises three to five carbon atoms (e.g., C₃-C₅alkyl). In other embodiments, the alkyl group is selected from methyl,ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl(n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl),1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl isattached to the rest of the molecule by a single bond. Unlessspecifically stated otherwise in the specification, an alkyl group isoptionally substituted by one or more of substituents. The term“alkenyl,” as used herein, denotes a monovalent group derived from ahydrocarbon moiety containing, in certain embodiments, from two to six,or two to eight carbon atoms having at least one carbon-carbon doublebond. The double bond may or may not be the point of attachment toanother group. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl,octenyl and the like.

The term “alkoxy” refers to a radical bonded through an oxygen atom ofthe formula —O-alkyl, where alkyl is an alkyl chain as defined above.

The term “pharmaceutically acceptable salt” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids includinginorganic or organic bases and inorganic or organic acids. Salts ofbasic compounds encompassed within the term “pharmaceutically acceptablesalt” refer to non-toxic salts of the compounds of this invention whichare generally prepared by reacting the free base with a suitable organicor inorganic acid. Representative salts of basic compounds of thepresent invention include, but are not limited to, the following:acetate, ascorbate, adipate, alginate, aspirate, benzenesulfonate,benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate,camphorate, camphorsulfonate, camsylate, carbonate, chloride,clavulanate, citrate, cyclopentane propionate, diethylacetic,digluconate, dihydrochloride, dodecylsulfanate, edetate, edisylate,estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate,glucoheptanoate, gluconate, glutamate, glycerophosphate,glycollylarsanilate, hemisulfate, heptanoate, hexanoate,hexylresorcinate, hydrabamate, bromide, chloride,2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isonicotinate,isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate,mesylate, methylnitrate, methylsulfate, methanesulfonate, mucate,2-naphthalenesulfonate, napsylate, nicotinate, nitrate, oleate, oxalate,pamoate (embonate), palmitate, pantothenate, pectinate, persulfate,phosphate/diphosphate, pimelate, phenylpropanoate, polygalacturonate,propionate, salicylate, stearate, sulfate, subacetate, succinate,tannate, tartrate, teoclate, thiocyanate, tosylate, triethiodide,trifluoroacetate, undeconate, valerate and the like. Furthermore, wherethe compounds of the invention carry an acidic moiety, suitablepharmaceutically acceptable salts thereof include, but are not limitedto, salts derived from inorganic bases including aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,mangamous, potassium, sodium, zinc, and the like. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, cyclic amines, dicyclohexylamines and basic ion-exchange resins, such as arginine, betaine,caffeine, choline, N,N-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like. Also, included are the basicnitrogen-containing groups that may be quaternized with such agents aslower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl;and diamyl sulfates, long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides, aralkyl halides like benzyland phenethyl bromides and others.

The term “subject” includes living organisms such as humans, monkeys,cows, sheep, horses, pigs, cattle, goats, dogs, cats, mice, rats,cultured cells, and transgenic species thereof. In a preferredembodiment, the subject is a human.

The term “administering” includes routes of administration which allowthe active ingredient of the invention to perform their intendedfunction.

The term “treat” or “treatment” refers to a method of reducing theeffects of a disease or condition. Treatment can also refer to a methodof reducing the underlying cause of the disease or condition itselfrather than just the symptoms. The treatment can be any reduction fromnative levels and can be, but is not limited to, the complete ablationof the disease, condition, or the symptoms of the disease or condition.

The term “inhibit,” “inhibition,” “inhibiting,” “prevent,” “prevention”or “preventing” means ameliorating, inhibiting or averting from symptomsassociated with the target disease or resulted by the target biologicalmechanism.

The term “cancer” or “cancer cell” refers to diseases in which abnormalcells divide without control and can invade nearby tissues, includingcarcinoma, sarcoma, leukemia, lymphoma and multiple myeloma, etc.Embodiments of a cancer include but are not limited to invasive breastcarcinoma, adenocarcinoma, lung cancer (non-small cell, squamous cellcarcinoma, adenocarcinoma, and large cell lung cancer), liver cancer,colorectal cancer, brain, head and neck cancer (e.g.,neuro/glioblastoma), breast cancer, ovarian cancer or carcinoma,transitional cell carcinoma of the bladder, prostate cancer, oralsquamous cell carcinoma, bone sarcoma, adrenocortical cancer,gastrointestinal tumors including colorectal cancer, biliary tractcancer such as gallbladder carcinoma (GBC), bladder cancer, esophagealcancer, gastric cancer, cervical cancer, salivary gland cancer, diarrheabenign neoplasm, ductal carcinoma in situ, paronychia,cholangiocarcinoma, kidney cancer, pancreatic cancer, medulloblastoma,glioblastoma, luminal, HER2-positive and triple negative mammary tumors,hematologic malignancies or leukemia (acute myelogenous leukemia (AML),B-precursor cell acute lymphoblastic leukemia (ALL), a fraction ofT-cell ALL, and chronic myelogenous leukemia (CML).

The phrase “therapeutically effective amount” refers to that amount of acompound, material, or composition comprising a compound of the presentinvention which is effective for producing a desired therapeutic effect,at a reasonable benefit/risk ratio applicable to any medical treatment.

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of the American ChemicalSociety or the Journal of Biological Chemistry. Specifically, butwithout limitation, the following abbreviations may be used in theexamples and throughout the specification: g (grams); mg (milligrams);mL (milliliters); μL (microliters); mM (millimolar); M (micromolar); Hz(Hertz); MHz (mega hertz); mmol (millimoles); hr or hrs (hours); min(minutes); MS (mass spectrometry); ESI (electrospray ionization); TLC(thin layer chromatography); and HPLC (high pressure liquidchromatography). For all of the following examples, standard work-up andpurification methods known to those skilled in the art can be utilized.Unless otherwise indicated, all temperatures are expressed in ° C.(degrees Centigrade). All reactions are conducted at room temperatureunless otherwise noted. Synthetic methodologies illustrated herein areintended to exemplify the applicable chemistry through the use ofspecific examples and are not indicative of the scope of the disclosure.

In particular, the present invention provides a compound of formula (I):

wherein R represents H or methoxy;R₁ and R₂ each independently selected from H, hydroxyl, halogen, C₁₋₃alkyl and C₁₋₃ alkoxy;R₃ to R₅ each independently selected from H, hydroxyl, fluoro, C₁₋₃alkyl and C₁₋₃ alkoxy;or a solvate, prodrug, stereoisomer, enantiomer, or pharmaceuticallyacceptable salt thereof.

In one embodiment, in formula (I), R is methoxy. In another embodiment,R₃ to R₅ each are H. In yet another embodiment, R₁ and R₂ each are H.

In one embodiment, in formula (I), R is H. In another embodiment, R₃ toR₅ each are H. In yet another embodiment, R₁ and R₂ each are H.

In a specific embodiment, in formula (I), R is methoxy, R₁ to R₅ eachare H, i.e., the compound 10-fluoro-2,3-dimethoxyrutaecarpine. Inanother specific embodiment, in formula (I), R and R₁ to R₅ each are H,i.e., the compound 10-fluoro-2-methoxyrutaecarpine (F-RUT).

The compounds of Formula (I) of the present invention are preparedaccording to general chemical synthetic procedures. The preparation ofthe embodiments of the compounds of the present invention is illustratedbelow. Suitable syntheses for compounds of the invention can be found inthe Examples below.

In one aspect, the compound of formula (I) is synthesized via theschemes below:

wherein condition (i) represents the presence of NaNO₂/HCl at 0° C.,condition (ii) represents the presence of acetic acid at 0° C.,condition (iii) represents the presence of formic acid at reflux andcondition (iv) represents the presence of SOCl₂/toluene.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of formula (I), or a pharmaceutically acceptableester, salt, or prodrug thereof, together with a pharmaceuticallyacceptable carrier.

To prepare the pharmaceutical compositions of this invention, one ormore compounds of the present invention as the active ingredient isintimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques, which carrier maytake a wide variety of forms depending of the form of preparationdesired for administration, e.g., oral or parenteral such asintramuscular. In preparing the compositions in oral dosage form, any ofthe usual pharmaceutical media may be employed. Thus, for liquid oralpreparations, such as for example, suspensions, elixirs and solutions,suitable carriers and additives include water, glycols, oils, alcohols,flavoring agents, preservatives, coloring agents and the like; for solidoral preparations such as, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches,sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit form, in which case solid pharmaceutical carriers areobviously employed. If desired, tablets may be sugar coated or entericcoated by standard techniques. For parenterals, the carrier will usuallycomprise sterile water, through other ingredients, for example, forpurposes such as aiding solubility or for preservation, may be included.Injectable suspensions may also be prepared, in which case appropriateliquid carriers, suspending agents and the like may be employed. Thepharmaceutical compositions herein will contain, per dosage unit, e.g.,tablet, capsule, powder, injection, teaspoonful and the like, an amountof the active ingredient necessary to deliver an effective dose asdescribed above.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude, aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles. Suitable dispersing or suspendingagents for aqueous suspensions, include synthetic and natural gums suchas tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin.

Tablets and capsules for oral administration are normally presented inunit dose form and contain conventional excipients such as binders,fillers (including cellulose, mannitol, lactose), diluents, tabletingagents, lubricants (including magnesium stearate), detergents,disintegrants (e.g. polyvinylpyrrolidone and starch derivatives such assodium glycolate starch), coloring agents, flavoring agents, and wettingagents (for example sodium lauryl sulfate).

The oral solid compositions can be prepared by conventional methods ofblending, filling or tableting. The blending operation can be repeatedto distribute the active principle throughout compositions containinglarge quantities of fillers. Such operations are conventional.

For parenteral administration fluid unit dosages can be prepared,containing the compound and a sterile vehicle. The compound can beeither suspended or dissolved, depending on the vehicle andconcentration. The parenteral solutions are normally prepared bydissolving the compound in a vehicle, sterilizing by filtration, fillingsuitable vials and sealing. Advantageously, adjuvants such as localanaesthetics, preservatives and buffering agents can also be dissolvedin the vehicle. To increase the stability, the composition can be frozenafter having filled the vials and removed the water under vacuum.Parenteral suspensions are prepared in substantially the same manner,except that the compound can be suspended in the vehicle instead ofbeing dissolved, and sterilized by exposure to ethylene oxide beforesuspending in the sterile vehicle. Advantageously, a surfactant orwetting agent can be included in the composition to facilitate uniformdistribution of the compound of the application.

Pharmaceutical preparation for administration by inhalation can bedelivered from an insufflator or a nebulizer pressurized pack.

In one aspect, the present invention provides a method of amelioratinginflammation in a subject, comprising a step of administering a compoundas disclosed herein to the subject.

In another aspect, the present invention provides a method ofsuppressing nitrogen oxide (NO) release in a subject, comprising a stepof administering a compound as disclosed herein to the subject.

In another aspect, the present invention provides a method ofsuppressing TNF-α release in a subject, comprising a step ofadministering a compound as disclosed herein to the subject.

In another aspect, the present invention provides a method of inhibitingcell migration, cell invasion or both in a subject, comprising a step ofadministering a compound as disclosed herein to the subject.

In another aspect, the present invention provides a method ofameliorating damage due to remodeling between the epithelium andendothelium in a subject, comprising a step of administering a compounddisclosed herein to the subject.

In another aspect, the present invention provides a method of reducingSnail Protein levels in a subject, comprising a step of administering acompound as disclosed herein to the subject.

In another aspect, the present invention provides a method of improvingcardiac, vasodilation and/or lung functions of a subject, comprising astep of administering a compound as disclosed herein to the subject.Preferably, the improvement comprises inhibition of fibrosis of heart,blood vessels and/or lung.

The compounds, or pharmaceutically acceptable salts thereof, areadministered orally, nasally, transdermally, pulmonary, inhalationally,buccally, sublingually, intraperintoneally, subcutaneously,intramuscularly, intravenously, rectally, intrapleurally, intrathecallyand parenterally. In one embodiment, the compound is administeredorally. One skilled in the art will recognize the advantages of certainroutes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

The invention having now been described by way of written description,those of skill in the art will recognize that the invention can bepracticed in a variety of embodiments and that the foregoing descriptionand examples below are for purposes of illustration and not limitationof the claims that follow.

EXAMPLES

All the key raw materials were purchased from various commercial sourcesand used without further purification. Some of the key raw materials andreagents were available in-house.

Synthetic Example 1

The compounds 10-fluoro-2-methoxyrutaecarpine (F-RUT) and10-fluoro-2,3-dimethoxyrutaecarpine are synthesized via the followingscheme:

Aniline (1) was subjected to the Sandmeyer reaction to give thediazonium salt, which was then coupled to a carboxylic acid (3) to yieldhydrazone (4), and treatment of the hydrazone (4) in an acidic conditiongave the carboline (5) in a 58% yield (three steps). The carboline (5)was then coupled with in-situ activated substituted o-aminobenzoic acidderivatives (6a,b), which were pretreated with thionyl chloride in thepresence of toluene at 70-80° C. to provide10-fluoro-2-methoxyrutaecarpine (F-RUT) and10-fluoro-2,3-dimethoxyrutaecarpine in 35% and 40% overall yields (foursteps), respectively.

The synthetic products were identified by ¹H and ¹³C nuclear magneticresonance (NMR), infrared (IR), and mass spectrometry (MS). For F-RUT,FT-IR (KBr, cm⁻¹): 3347 (N−H) and 1652 (carbonyl group). ¹H-NMR (CDCl₃,ppm): δ 3.18 (t, J=6.9 Hz, 2H, 2H-8), 4.57 (t, J=6.9 Hz, 2H, H−7), 3.93(s, 3H, 2-OMe), 7.02 (dd, J=8.9, 2.4, 1H, H−3), 7.05 (d, J=2.4, 1H,H−1), 8.21 (d, J=8.9, 1H, H−4), 7.11 (d, J=8.8, 1H, H−12), 7.38 (d,J=8.8, 1H, H−11), 8.99 (s, 1H, H−9), 12.04 (s, 1H, N−H). MS-ESI (m/z)([M-H]⁻): calcd. 335.3; found 334.4. For10-fluoro-2,3-dimethoxyrutaecarpine (F_(2MO)-RUT), FT-IR (KBr, cm⁻¹):3398 (N−H) and 1641 (carbonyl group). ¹H-NMR (CDCl₃, ppm): δ 3.18 (t,J=6.8 Hz, 2H, H−8), 4.57 (t, J=6.8 Hz, 2H, H−7), 3.92 (s, 3H, O-Me),3.88 (s, 3H, O-Me), 7.04 (s, 1H, H−1), 7.11 (d, J=8.9, 1H, H−12), 7.37(d, J=8.9, 1H, H−11), 7.66 (s, 1H, H−4), 8.94 (s, 1H, H−9), 11.87 (s,1H, N−H). MS-ESI (m/z) ([M-H]⁻): calcd. 365.3; found 364.4.

Biological Assay

Preparation Example 2—Cell Culture

The RAW264.7 macrophage cell line and A2780 ovarian carcinoma cells weregrown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetalbovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mMsodium pyruvate, 4.5 g/L glucose, 4 mM 1-glutamine, and 1.5 g/L sodiumbicarbonate at 37° C. in a humidified atmosphere with 5% CO2. Primaryhuman aortic ECs (HAECs) were grown in a MesoEndo Endothelial CellGrowth Medium Kit (Cell Applications, San Diego, Calif., USA)supplemented with 10% FBS at 37° C. in a humidified atmosphere with 5%CO2.

Example 3—Suppression of NO and TNF-Alpha Releases by LPS-Treated Cells

NO production was evaluated by measuring the nitrite concentration insupernatants of cultured RAW264.7 macrophages. Cells were first seededat a density of 2×10⁵ cells/mL in 24-well plates for 24 h, followed byco-treatment with different concentrations of F-RUT withlipopolysaccharide (LPS) (40 ng/mL) for another 24 h. The amount ofnitrite in cell culture supernatants was detected using the Griessreagent (1% sulfanilamide in 5% phosphoric acid and 0.1%naphthylethylenediamine dihydrochloride in water). Data are reported asthe mean±standard error of the mean (SEM) values of three independentdeterminations.

NO production of LPS-treated RAW264.7 macrophages increased compared tothat of untreated cells. Co-treatment with the synthesized F-RUTsuppressed NO production in a concentration-dependent (0-20 μM) manner(*p<0.05, **p<0.01, compared to the LPS-treated group) (FIG. 1(a)). Aconsistent concentration-dependently potent (*p<0.05, compared to theLPS-treated group) (FIG. 1(b)) suppressive effect of TNF-α released intothe medium was also shown. The suppressive effects were not due tocytotoxic activity because F-RUT showed substantially no cytotoxicity toRAW264.7, H460 and CL1-3 cells at concentrations of 0-20 μM (*p<0.05,**p<0.01, compared to the RUT-treated group) (FIG. 1(c)).

Example 4—Suppression of Inducible (i)NOS and COX-2 Expressions

An MTT assay to test cell viability was performed based on theconversion of the yellow tetrazolium salt to the purple formazanproduct. Cells (104 cells/well) were grown in a 96-well platesupplemented with standard culture medium. Cells were treated with RUTand F-RUT (0-20 μM) for 24 h. An MTT stock solution (5 mg of MTT/mL ofphosphate-buffered saline; PBS) was added to the growing cultures for 2h. The absorbance was measured with a spectrophotometer at 560 nm. DMSOalone was measured as a reading control. Data were reported as themean±SEM values of five independent determinations.

Protein samples were separated and resolved by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferredonto a polyvinylidene difluoride (PVDF) membrane. The membrane wasincubated with a primary antibody at 4° C. overnight, and then incubatedwith a horseradish peroxidase (HRP)-conjugated secondary immunoglobulinG (IgG) antibody; immunoreactive bands were visualized with PerkinElmerenhanced chemiluminescent reagents.

RAW264.7 macrophages were seeded in a 96-well plate with DMEM. Then,cells were transfected with the pGL4.32 [luc2P/NF-κB-RE/Hygro] (Promega,Madison, Wis., USA) plasmid reporter gene using TurboFect TransfectionReagent (Fermentas, Glen Burnie, Md., USA). At 24 h after transfection,cells were treated with LPS (40 ng/mL) and F-RUT for 24 h in serum-freemedium. Then the luciferase activity was detected by the luminescencemeasured in a luminescence microplate reader (Thermo Varioskan Flash,Waltham, Mass., USA) using a ONE-Glo luciferase assay kit (Promega).Luciferase activities were normalized to protein concentrations.

The inventors observed that LPS-treated RAW264.7 macrophages exhibitedsignificantly elevated protein amounts of iNOS and COX-2, while F-RUTsuppressed their expressions in a concentration-dependent manner (FIG.2(a)). 3-Actin protein levels of the loading controls remained constant.In the inflammation reaction, NF-κB activation triggers the induction ofCOX-2 and iNOS. We determined whether F-RUT suppressed NF-κB activationin LPS-activated macrophages. An NF-κB-dependent luciferase reporterplasmid was transiently transfected in LPS-induced macrophages toconfirm whether F-RUT inhibited NF-κB-binding activity. F-RUT inhibitedLPS-induced NF-κB transcriptional activity at 0-2.5 μM (*p<0.05,**p<0.01, compared to the LPS-treated group) (FIG. 2(b)). The resultssuggested that inhibition of iNOS and COX-2 expression by F-RUT wascorrelated with suppression of NF-κB activation. The inventors alsoobserved that, in addition to COX-2 suppression, the enzyme activity isalso inhibited. Compared to RUT, F-RUT showed less cytotoxicity, butretained the anti-inflammatory activity.

Example 5—Inhibition of Cell Migration/Invasion

A2780 cells were cultured at a density of 2×10⁵ cells/well in 6-wellplates and incubated at 37° C. for 24 h. A centerline in the cells wasscratched using a 200-μL pipette tip and washed with PBS. Then, newcomplete medium was added and treated with or without 1 and 2.5 μM ofF-RUT for 24 h. At the endpoint of incubation, cells were examined andphotographed with an optical microscope. The distance between the edgesof the scratched area was measured and calculated to estimate themigratory ability of cells.

A2780 cell invasion was evaluated using 24-well transwell inserts(8-μm-pore filters, Merck Millipore) individually coated with Matrigel(BD Biosciences, Bedford, Mass., USA). A2780 cells (2×104 cells in eachwell) were cultured for 24 h with serum-free minimum essential medium(MEM) and then treated with F-RUT (1.25 or 2.5 μM) for another 24 h inthe upper chamber of the transwell. Medium containing 10% FBS was placedin the lower chamber. At the end of incubation, non-migrated cells wereremoved using a cotton swab; cells that had penetrated to the oppositesurface of the filter were fixed with 4% formaldehyde and stained with2% crystal violet. Stained cells were counted and photographed under aphase-contract optical microscope at 200× magnification. Threeindependent experiments were performed as described elsewhere.

As illustrated in FIG. 3(a), wound-healing assays used an ovariancarcinoma A2780 cell line in the presence of F-RUT (0-5 μM) for 0-24 h.The migration rate was measured using imaging software, and Student'st-test was used for the statistical analysis. F-RUT showed significanteffects against cell migration. F-RUT (0-2.5 μM) treatments for 24 halso exhibited invasion inhibitory activity in a transwell assay (FIG.3(b)).

Example 6—Activation of TRPV1 and eNOS

TRPV1 is reportedly present in ECs of arteries. To validate theexpression of TRPV1 in the endothelium, the TRPV1 protein of humanaortic ECs (HAECs) was detected using immunoblotting; the method ofWestern Blot Analysis has been stated in Example 4. F-RUT treatment (20μM) for 15 min increased TRPV1 protein amounts two-fold compared to thecontrol group after normalization with α-tubulin levels (FIG. 4). Theeffect of F-RUT on the phosphorylation of eNOS in HAECs is furtherexamined because NO production is consequently regulated by thephosphorylation of eNOS. F-RUT treatment (20 μM) for 15 minsignificantly increased the phosphorylation of eNOS 1.4-fold compared tothe control group after normalization with total eNOS (lower panel).F-RUT upregulated the expression of TRPV1 and activated eNOSphosphorylation in ECs.

Animal Experiment

Example 7—Amelioration of Inflammation in OVA/Alum-Challenged Mice

BALB/c mice (six weeks of age) were obtained from the Animal Center ofthe College of Medicine, National Taiwan University (Taipei, Taiwan),and sensitized with an intraperitoneal injection of 20 μg of ovalbumin(OVA) emulsified in 2 mg of aluminum hydroxide in a total volume of 200μL phosphate-buffered saline (PBS) on day 0, and boosted with 50 μg ofOVA emulsified in 4 mg of aluminum hydroxide on days 14 and 28. RUT orF-RUT was given by oral administration on days 30, 32, 34, 36, and 38.For post-challenge, all mice were treated intranasally with OVA (100 μgin a total volume of 40 μL PBS) on days 40, 41, 42, and 43. At 24 hafter the last OVA challenge, mice were sacrificed, and their organswere collected. All experimental procedures were reviewed and approvedby the Institutional Animal Care and Use Committee or Panel. Lungtissues were fixed in 4% paraformaldehyde (sc-281692; SantaBiotechnology) and embedded in paraffin. Tissue sections were made at a5-μm thickness, and stained with hematoxylin and eosin (H&E) solutionfor examination of inflammation.

There was predominant inflammation in the lungs accomplished byincreased infiltrating neutrophils after mice had been challenged withOVA/alum for 44 days. Alternate-day oral administration of RUT or F-RUTameliorated the OVA/alum-induced lung inflammation and showed a similarpattern to the untreated control group (FIG. 5(a)).

KLF10 gene is regulated by transforming growth factor (TGF)-β/Smad.Deletion of Klf10 in mice is associated with significant inflammation ofthe lungs challenged with OVA/alum for 44 days. In another experiment,KLF-10 KO mice was used. There was predominant inflammation in the micelungs accomplished by increased infiltrating neutrophils afterchallenged with OVA/alum for 44 days. Alternate-day oral administrationof F-RUT ameliorated the OVA/alum-induced lung inflammation (FIG. 5(b)).The collagen formation around the bronchus was decreased after the oraladministration of F-RUT (25% in OVA/Alum group, 18% in F-RUT group)(FIG.5(c)). These results suggest the beneficial effect of F-RUT oninflammation induced fibrosis.

Example 8—Amelioration of Inflammation-Stimulated Respiratory Interfacein KLF10 KO Mice

Blood-Air-Barrier (alveolar-capillary membrane), gas exchanging regionof the lungs, was measured to perform the lung function. After theKLF-10 KO mice were challenged with OVA/Alum and oral administration ofRUT or F-RUT, the lung was harvested to measure the thickness ofblood-air-barrier by TEM. The average thickness of blood-air-barrier wassignificant increased in OVA/Alum group (586 nm compared to 348 nm incontrol group) and reversed by RUT or F-RUT (418 and 393 nm,respectively), see FIG. 6. These data explored the potential biologicalfunction of F-RUT in protecting lung from inflammation.

Example 9—Inhibition of ROS Activity in Zebrafish

FIG. 7 shows that the anti-inflammation effects of F-RUT not only incell but also in zebrafish model. LPS significantly induced ROS level inzebrafish at 10 and 20 ng/ml (***p<0.001, compared to control group). 5ng/ml F-RUT suppressed the LPS-induced ROS as similar level to the grouptreated with F-RUT only (***p<0.001, compared to LPS group).

The inventors find the following phenomena to evidence the claimedeffect.

First, vasodilator effects of RUT to induce CGRP synthesis and releasewere via activation of TRPV1. Therefore, RUT's analogs were designed andsynthesized for better vasodilator effects. Structural modifications ofRUT were designed to enhance its biological activities. However,increased cytotoxicity hampers their application in vascular disorders.Fluorinated RUT, novel analogs provided herein with very lowcytotoxicity, showed anti-inflammatory activity (Examples 7 to 9) andmigration/invasion-suppressive activities (Example 5) that arebeneficial in reducing side effects when used for pharmaceutics. Inaddition, eNOS and iNOS are isoforms with identical promoter elementsthat drive similar biological effects. With respect to the diverseeffects of fluorinated RUT on eNOS and iNOS described herein, they couldhave resulted from different signaling pathways in macrophages and ECs.Fluorinated RUT suppressed iNOS in macrophages, while it activated eNOSin Ecs (Examples 4 and 6). The results bolster Fluorinated RUT, derivedfrom RUT, having enhanced beneficial effects and reduced adverseeffects.

OVA/alum-sensitized mice are a well-known animal model to induce lunginflammation. In the airways, there are increased granulocytes, forexample, neutrophils, and remodeling of the interstitium (capillaryendothelium, alveolar epithelium, basement membrane, and perivasculartissue). Here, the examples showed that fluorinated RUT reducedinfiltrating neutrophils and maintained the air sac structure inOVA/alum-challenged mice (Example 7). These results might imply not justan anti-inflammatory effect but its benefit against damage due toremodeling between the epithelium and endothelium as well.

Hypertension activates pro-oxidant enzymes resulting in increased ROSformation, which is associated with Ang-II and mechanical forces, anddamage to the vasculature. Inflammation, migration, and fibrosis areimportant factors contributing to endothelial dysfunction andcardiovascular remodeling. Oxidative stress plays a physiological rolein controlling endothelial function and also a pathophysiological role.Many heart injuries result in fibrosis with deposition of excesscollagens, or other matrix proteins, leading to the development of heartfailure. Inflammation is the initial and primary trigger in cardiacstress and involves elevated levels of inflammatory cytokines andchemokines in tissues. Fibrosis is characterized by the excessproduction of ECM produced by myofibroblasts. Cardiac fibroblastsoriginate from the endothelial-to-mesenchymal transition (EndMT) of ECs,which is important in the formation of cardiac fibroblasts. The EndMT isregulated by signaling pathways mediated by inflammation-associatedcytokines. Direct contact with the bloodstream makes the endothelium apromising target for drug treatment. Ischemia/reperfusion injury leadingto cardiac fibrosis is mainly mediated by collagen deposition bymyofibroblasts. Snail induction is involved in fibrosis when undergoingthe EndMT. Snail inhibitors remarkably suppressed collagen depositionand cardiac fibrosis in mice. Fluorinated RUT treatment of A2780 cellsproduced reduced Snail protein levels, which suggests that inhibition ofthe EndMT by fluorinated-RUT could be a new strategy for combatingvascular diseases (figure not shown).

A previous study illustrated that inflammation and myofibroblastformation contribute to the development of pulmonary fibrosis.Inflammatory cytokines induce the transformation of ECs tomyofibroblasts through the EMT, and then produce excess ECM causingfibrosis. Fluorinated-RUT, a class of RUT derivatives, possesses lowcytotoxicity but retains its activities against inflammation andmigration/invasion. Treatment with fluorinated RUT enhanced TRPV1 andactivated eNOS activity. According to the examples provided above,fluorinated RUT would provide applications in improving cardiac,vasodilation, and lung functions.

What is claimed is:
 1. A compound having the following Formula (I),

wherein R represents H or methoxy; R₁ and R₂ are each independentlyselected from H, hydroxyl, halogen, C₁₋₃ alkyl and C₁₋₃ alkoxy; R₃, R₄,and R₅ are each independently selected from H, hydroxyl, fluoro, C₁₋₃alkyl and C₁₋₃ alkoxy; or a solvate, prodrug, stereoisomer, enantiomer,or pharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein R is methoxy.
 3. The compound of claim 2, wherein R₃, R₄, and R₅are each H.
 4. The compound of claim 3, wherein R₁ and R₂ each are H. 5.The compound of claim 1, wherein R is H.
 6. The compound of claim 5,wherein R₃, R₄, and R₅ are each H.
 7. The compound of claim 6, whereinR₁ and R₂ each are H.
 8. A pharmaceutical composition comprising acompound of claim 1, or a pharmaceutically acceptable ester, salt, orprodrug thereof, together with a pharmaceutically acceptable carrier. 9.A method of ameliorating inflammation in a subject, comprising a step ofadministering a compound of claim 1 to the subject.
 10. A method ofameliorating damage due to remodeling between the epithelium andendothelium in a subject, comprising a step of administering a compoundof claim 1 to the subject.
 11. A compound of10-fluoro-2-methoxyrutaecarpine (F-RUT).
 12. A compound of10-fluoro-2,3-dimethoxyrutaecarpine.